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SummaryAcute stroke causes substantial mortality and morbidity and provokes extensive changes to myeloid immune cell populations in the brain that may be targets for limiting brain damage and enhancing brain repair. The most effective immunomodulatory approaches will require precise manipulation of discrete myeloid cell phenotypes in time and space to avoid harmful effects of indiscriminate neuroimmune perturbation. We sought to define how stroke alters the composition and phenotypes of mononuclear myeloid cells with particular attention to how cell ontogeny and spatial organisation combine to expand myeloid cell diversity across the brain after stroke. Multiple reactive microglial states and dual monocyte-derived populations contributed to an extensive repertoire of myeloid cells in post-stroke brain. We identified important overlap and distinctions among different cell types and states that involved ontogeny- and spatial-related properties. Notably, brain connectivity with infarcted tissue underpinned the pattern of local and remote altered cell accumulation and reactivity. Our discoveries suggest a global but anatomically-governed brain myeloid cell response to stroke that comprises diverse phenotypes arising through intrinsic cell ontogeny factors interacting with exposure to spatially-organised brain damage and neuroaxonal cues.

Microglial endolysosomal (dys)function is strongly implicated in neurodegeneration. Transcriptomic studies show that a microglial state characterised by a set of genes involved in endolysosomal function is induced in both mouse Alzheimer’s Disease (AD) models and in human AD brain and that the onset of this state is emphasized in females.Cst7(encoding protein Cystatin F) is among the most highly upregulated genes in these microglia. However, despite such striking and robust upregulation, the sex-specific function ofCst7in neurodegenerative disease is not understood. Here, we crossedCst7−/−mice with theAppNL-G-Fmouse to test the role ofCst7in a model of amyloid-driven AD. Surprisingly, we found thatCst7plays a sexually dimorphic role regulating microglia in this model. In females,Cst7-deficient microglia had greater endolysosomal gene expression, lysosomal burden, and amyloid beta (Aβ) burdenin vivoand were more phagocyticin vitro. However, in males,Cst7-deficient microglia were less inflammatory and had a reduction in lysosomal burden but had no change in Aβ burden. This study has important implications for AD research, confirming the functional role of a gene which is commonly upregulated in disease models, but also raising crucial questions on sexual dimorphism in neurodegenerative disease and the interplay between endolysosomal and inflammatory pathways in AD pathology.

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterize pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus, and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted p < 0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

AimsPharmacological activation of the antioxidative transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) improves outcomes in experimental models of intracerebral haemorrhage (ICH). However, the Nrf2 pathway has not been previously studied in humans after ICH. Our study aims to address this gap.MethodsWe selected cases with fatal ICH from a prospective community-based inception cohort study and age-matched and sex-matched controls who died suddenly of non-neurological disease. We used immunohistochemistry to quantify Nrf2 (% total area stained overall and % of nuclei stained) and CD68 expression in controls and perihaematomal, ipsilateral and contralateral brain tissue from cases. We measured downstream haem oxygenase-1 (HMOX1) and NAD(P)H dehydrogenase quinone 1 [NQO1] expression using RNA in situ hybridisation.Results26 ICH cases (median age: 82 (IQR 76–86); 13 (50%) male) and eight controls (median age: 79 (IQR 77–80); 3 (37.5%) male) were included. We found no significant differences in overall % of Nrf2 staining between ICH cases and controls. However, the mean % of nuclei staining for Nrf2 seemed higher in perihaematomal compared with contralateral regions, although this was only statistically significant >60 days after ICH (25% (95% CI 17% to 33%) vs 14% (95% CI 11% to 17%), p=0.029). The percentage of perihaematomal tissue staining for CD68 was higher >60 days after ICH (6.75%, 95% CI 2.78% to 10.73%) compared with contralateral tissue (1.45%, 95% CI 0.93% to 1.96%, p=0.027) and controls (1.08%, 95% CI 0.20% to 1.97%, p=0.0008). RNA in situ hybridisation suggested increased abundance of HMOX1 and NQO1 transcripts in perihaematomal versus distant ipsilateral brain tissue obtained ConclusionsWe found evidence of Nrf2 activation in human brain tissue after ICH. Pharmacological augmentation of Nrf2 activation after ICH might be a promising therapeutic approach.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. [Methods] Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. [Results] Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (K [min]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. [Discussion] Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules., The authors acknowledge Lipoid Co. (Germany) for the lipid sample gifts, the staff at the bioimaging facility at the University of Manchester and NTU for advice on confocal imaging and data analysis. The authors also would like to acknowledge the access to the Leica Thunder Imager 3D Cell Culture system funded by the Office for Students at NTU. They also wish to thank the Biological Services Facility at the University of Manchester for the excellent animal husbandry. The cost of this study has been covered by: a) Dr Z Al-Ahmady research fellowship (School of Pharmacy and Optometry, University of Manchester), b) QR fund and Vice-Chancellor PhD studentship awarded for Dr Zahraa Al-Ahmady and Satinderdeep Kaur, Nottingham Trent University, c) BBSRC DTP Studentship awarded to Jessica Bates, Nottingham Trent University, d) MRC DTP studentship to Jack Barrington at University of Manchester. Jack Barrington also received support from the Natalie Kate Moss Trust, e) MRC grant (MR/N003586/1) for Eloise Lemarchand at University of Manchester, and f) EPSRC grant for Dr Ben Dickie at the University of Manchester (EP/S021510). Graphical abstract and schematic graphes were performed using Biorender.

Background: The ‘Sepsis Six’ bundle was promoted as a more deliverable tool outside of the critical care settings, but there is very little data available on the progress and change of sepsis care outside the critical care environment in the UK. Our aim was to compare the yearly prevalence, outcome and the Sepsis Six bundle compliance in patients at risk of mortality from sepsis in non-intensive care environments. Methods: Patients with a National Early Warning Score (NEWS) of 3 or above and suspected or proven infection were enrolled into four yearly 24-hour point prevalence studies, carried out in fourteen hospitals across Wales from 2016-2019. Results: Out of the 26,947 patients screened 1,651 fulfilled inclusion criteria and were recruited. The full ‘Sepsis Six’ care bundle was completed on 223 (14.0%) occasions, with no significant difference between the years. On 190 (11.5%) occasions none of the bundle elements were completed. There was no significant correlation between bundle element compliance, NEWS or year of study. One hundred and seventy (10.7%) patients were seen by critical care outreach; the ‘Sepsis Six’ bundle was completed significantly more often in this group (54/170, 32.0%) than for patients who were not reviewed by critical care outreach (168/1385, 11.6%; pConclusions: Our data suggests that despite efforts to increase sepsis awareness within the NHS, there is poor compliance with the sepsis care bundles and no change in the high mortality over the study period. Further research is needed to determine which time-sensitive ward-based interventions can reduce mortality in patients with sepsis and how can these results be embedded to routine clinical practice.Trial registration: Defining Sepsis on the Wards ISRCTN 86502304 https://doi.org/10.1186/ISRCTN86502304 prospectively registered 09/05/2016

Microglia are tissue-resident macrophages playing essential roles in central nervous system (CNS) development and homeostasis [14, 17]. The importance of microglia for brain health in humans has been highlighted by the definition of Mendelian disorders associated with dysfunction of microglia-related proteins. These so-called microgliopathies [20] comprise a diverse set of neurological phenotypes including disease due to mutations in CSF1R [5, 8, 13], DAP12 and TYROBP/TREM2 [4, 7], USP18 [3, 11, 16, 18], and IRF8 [2, 6]. Here, we describe a novel early onset lethal encephalopathy due to mutations in the microglial-associated protein NRROS.

Background Mesenchymal stem cells (MSCs) hold great potential as a therapy for stroke and have previously been shown to promote recovery in preclinical models of cerebral ischaemia. MSCs secrete a wide range of growth factors, chemokines, cytokines and extracellular vesicles—collectively termed the secretome. In this study, we assessed for the first time the efficacy of the IL-1α-primed MSC-derived secretome on brain injury and functional recovery after cerebral ischaemia. Methods Stroke was induced in male C57BL/6 mice using the intraluminal filament model of middle cerebral artery occlusion. Conditioned medium from IL-1α-primed MSCs or vehicle was administered at the time of reperfusion or at 24 h post-stroke by subcutaneous injection. Results IL-1α-primed MSC-derived conditioned medium treatment at the time of stroke led to a ~ 30% reduction in lesion volume at 48 h and was associated with modest improvements in body mass gain, 28-point neurological score and nest building. Administration of MSC-derived conditioned medium at 24 h post-stroke led to improved nest building and neurological score despite no observed differences in lesion volume at day 2 post-stroke. Conclusions Our results show for the first time that the administration of conditioned medium from IL-1α-primed MSCs leads to improvements in behavioural outcomes independently of neuroprotection.

BACKGROUND AND PURPOSE Studies in animal models suggest that inflammation is a major contributor to secondary injury after intracerebral hemorrhage (ICH). Direct, noninvasive monitoring of inflammation in the human brain after ICH will facilitate early-phase development of anti-inflammatory treatments. We sought to investigate the feasibility of multimodality brain imaging in subacute ICH. METHODS Acute ICH patients were recruited to undergo multiparametric MRI (including dynamic contrast-enhanced measurement of blood-brain barrier transfer constant (Ktrans) and PET with [11C]-(R)-PK11195). [11C]-(R)-PK11195 binds to the translocator protein 18 kDa (TSPO), which is rapidly upregulated in activated microglia. Circulating inflammatory markers were measured at the time of PET. RESULTS Five patients were recruited to this feasibility study with imaging between 5 and 16 days after onset. Etiologies included hypertension-related small vessel disease, cerebral amyloid angiopathy (CAA), cavernoma, and arteriovenous malformation (AVM). [11C]-(R)-PK11195 binding was low in all hematomas and 2 (patient 2 [probable CAA] and 4 [AVM]) cases showed widespread increase in binding in the perihematomal region versus contralateral. All had increased Ktrans in the perihematomal region (mean difference = 2.2 × 10−3 minute−1; SD = 1.6 × 10−3 minute−1) versus contralateral. Two cases (patients 1 [cavernoma] and 4 [AVM]) had delayed surgery (3 and 12 months post-onset, respectively) with biopsies showing intense microglial activation in perilesional tissue. CONCLUSIONS Our study demonstrates for the first time the feasibility of performing complex multimodality brain imaging for noninvasive monitoring of neuroinflammation for this severe stroke subtype.

Stroke is one of the leading causes of death and disability worldwide. Inflammation plays a key role across the time course of stroke, from onset to the post-injury reparative phase days to months later. Several regulatory molecules are implicated in inflammation, but the most established inflammatory mediator of acute brain injury is the cytokine interleukin-1. Interleukin-1 is regulated by large, macromolecular complexes called inflammasomes, which play a central role in cytokine release and cell death. In this review we highlight recent advances in inflammasome research and propose key roles for inflammasome components in the progression of stroke damage.

SummarySynapse loss correlates with cognitive decline in Alzheimer’s disease (AD). Data from mouse models suggests microglia are important for synapse degeneration, but direct human evidence for any glial involvement in synapse removal in human AD remains to be established. Here we observe astrocytes and microglia from human brains contain greater amounts of synaptic protein in AD compared to non-disease controls, and that proximity to amyloid-β plaques and theAPOE4risk gene exacerbate this effect. In culture, mouse and human astrocytes and primary mouse and human microglia phagocytose AD patient-derived synapses more than synapses from controls. Inhibiting MFG-E8 function rescued the elevated engulfment of AD synapses by astrocytes and microglia without affecting control synapse uptake. Thus, AD promotes increased synapse ingestion by human glial cells via an MFG-E8 opsonophagocytic mechanism with potential for targeted therapeutic manipulation.One-Sentence SummaryGlial cells ingest synapses in Alzheimer’s disease and antibody treatment reduces this ingestion in cultured human cells.

Cerebral malaria (CM) is a serious neurological complication caused by Plasmodium falciparum infection. Currently, the only treatment for CM is the provision of antimalarial drugs; however, such treatment by itself often fails to prevent death or development of neurological sequelae. To identify potential improved treatments for CM, we performed a nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental CM (ECM). Bioinformatics analyses revealed IL33 as a critical regulator of neuroinflammation and cerebral pathology that is down-regulated in the brain during fatal ECM and in the acute period following treatment of ECM. Consistent with this, administration of IL33 alongside antimalarial drugs significantly improved the treatment success of established ECM. Mechanistically, IL33 treatment reduced inflammasome activation and IL1β production in microglia and intracerebral monocytes in the acute recovery period following treatment of ECM. Moreover, treatment with the NLRP3-inflammasome inhibitor MCC950 alongside antimalarial drugs phenocopied the protective effect of IL33 therapy in improving the recovery from established ECM. We further showed that IL1β release from macrophages was stimulated by hemozoin and antimalarial drugs and that this was inhibited by MCC950. Our results therefore demonstrate that manipulation of the IL33–NLRP3 axis may be an effective therapy to suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of CM.

Studies in animal models suggest that inflammation is a major contributor to secondary injury after intracerebral hemorrhage (ICH). Direct, noninvasive monitoring of inflammation in the human brain after ICH will facilitate early-phase development of anti-inflammatory treatments. We sought to investigate the feasibility of multimodality brain imaging in subacute ICH.Acute ICH patients were recruited to undergo multiparametric MRI (including dynamic contrast-enhanced measurement of blood-brain barrier transfer constant (KFive patients were recruited to this feasibility study with imaging between 5 and 16 days after onset. Etiologies included hypertension-related small vessel disease, cerebral amyloid angiopathy (CAA), cavernoma, and arteriovenous malformation (AVM). [Our study demonstrates for the first time the feasibility of performing complex multimodality brain imaging for noninvasive monitoring of neuroinflammation for this severe stroke subtype.

Stroke is one of the leading causes of death and disability worldwide. Inflammation plays a key role across the time course of stroke, from onset to the post‐injury reparative phase days to months later. Several regulatory molecules are implicated in inflammation, but the most established inflammatory mediator of acute brain injury is the cytokine interleukin‐1. Interleukin‐1 is regulated by large, macromolecular complexes called inflammasomes, which play a central role in cytokine release and cell death. In this review we highlight recent advances in inflammasome research and propose key roles for inflammasome components in the progression of stroke damage.